EMBRYOLOGY OF CRYPTOBRANCHUS 95 



rapidity, an important consideration when great series of large 

 sections are to be handled in considerable numbers. 



In sectioning and staining the early cleavage stages the exact 

 mode of procedure is as follows: 



From formalin pass the eggs to alcohol, 35 per cent, 50 per cent, two hours each. 



Grenacher's borax carmine in 70 per cent alcohol, about two days. 



Acid alcohol (0.25 per cent HCl in 70 per cent alcohol), about two hours. 



Ninety-five per cent alcohol, two to twelve hours; 100 per cent alcohol, two to 

 three hours. 



Xylol, four to ten hours. 



Paraffin with melting point 52° C. (at a temperature not exceeding 55° C), two 

 days. Change the paraffin at least once. 



Imbed in a paper box, hardening the block under alcohol. 



Cut sections 10m to 15m thick, using a Minot rotary microtome. 



Counterstain on the slide with Lyons blue and picric acid mixture in absolute 

 alcohol. 



Wash in xylol long enough to destain slightly. 



Mount in- Canada balsam. 



Early stages require longer for fluids (especially paraffin) to 

 penetrate than do later stages. For the yolk-laden ovarian eggs, 

 and maturation and fertilization stages, from two to three days 

 in borax carmine, and about three days in melted paraffin, are 

 necessary. I have found no serious ill effects in these stages from 

 this prolonged immersion in paraffin at the temperature given. 



Material fixed in Lavdowsky's solution stains and infiltrates 

 more rapidly than with the other methods of fixation; also the 

 yolk ig less likely to crumble. 



Late cleavage, and gastrula stages, are penetrated by the vari- 

 ous fluids more rapidly than the early cleavage stages, so that the 

 time may be reduced to two-thirds or one half. For still later 

 stages, there is a further gradual reduction in the length of time 

 required. 



During the present year, at the suggestion of Professor Wilson, 

 I have employed a shght modification of the method described 

 above. After being cleared in xylol, the objects were left several 

 days in a mixture of xylol and paraffin at about 38° C. By this 

 preliminary treatment, the time required for infiltration with 

 melted paraffin at a high temperature was reduced at least oae- 

 half, with some improvement in the quality of the preparations. 



